Sequencing fragments of cryptophyte plastomes from 16S rRNA to rbcL genes and phylogenetic analyses based on the protein-encoding genes located in these fragments

In recent phylogenetic analyses combining nuclear and nucleomorph RNA genes of the ribosomal operons, three different colourless lineages were found in the genus Cryptomonas. This raised questions about the evolutionary history of these interesting objects and their relatives as well as the role of plastid genomes such as whether these three lineages resulted from similar or from different evolutionary events or what are the mutual relationship or/and roles of photosynthetic genes in the absence of photosynthetic activities, etc. To answer the interesting questions, the biological information has to been collected systematically from their plastid genomes. At the first stage of the thesis, the cryptophyte plastid rbcL gene (1,5- biphosphate carboxylase/oxygenase [RuBisCO] large subunit) was chosen to amplify by BioTherm� Taq DNA Polymerase and read their DNA compositions by SequiTherm EXCEL� II DNA Sequencing Kit-LC and Li-Cor 4200L bidirectional sequencer. Eighteen newly rbcL sequences of Cryptomonas strains were obtained. Of these, five sequences were from heterotrophic (colorless) strains and the remaining was from photosynthetic (pigmented) strains. The results of rbcL phylogeny analyses showed that the colorless C. paramecium and their closely relative photosynthetic Cryptomonas had increased their evolutionary rates significantly. These were congruent with those of nuclear rDNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA) and nucleomorph SSU rDNA that had been examined in previously. They were combined with other result done by Dr. Kerstin Hoef-Emden such as analyzing the shift from NNC to NNU in two-fold degenerate NNY codon in rbcL gene in Cryptomonas to discuss some hypotheses of the loss of photosynthetic activities in the colorless C. paramaecium strains. Detail results and discussion were published in BMC Evolutionary Biology 2005; 5:56. In the second part of the thesis, the goals were to amplify the cryptophyte plastome 16S rRNA-rbcL fragments by MasterAmpTM Extra-long PCR kit and read their DNA sequences by BigDye Terminator v1.1 Cycle sequencing kit and automated ABI3730 sequencer, then exploited the sequencing information for further understanding the evolutionary history of cryptophyte plastomes. The task also attempted to find new evidence to explain the relationship between the changing from autotrophic to heterotrophic lifestyle in colorless Cryptomonas lineages and the elevation of evolutionary rates of photosynthetic genes that were located in the plastome 16S rRNA-rbcL fragment. Twenty-two cryptophyte strains (four of them were colorless) were participated in this part. Most of the fragments (15) were read completely as planned while several fragments (7) were not, due to lack of time. The colorless strains possessed the smallest fragments; their plastomes, thus, were predicted to be the smallest among those of Cryptomonas. Strain C. erosa CCAC 0018 and C. obovoidea CCAC 0031 seemed to have the largest plastomes as their 16S-rbcL fragments contained an additional gene � ycf26 � that was not found in other Cryptomonas strains. Advantages and disadvantages of long-range PCR and primer-walking sequencing combination were discussed. Based on the conserved domain analyses, all ycf26 from secondary plastids seems to be inactive and on the way to become pseudogene than alter its function. Another additional gene � ORF403 encoding Tic22 protein � also was examined the conserved domains and done a phylogenetic analysis. Some specific characteristics of ORF403 in rhodoplasts and cryptophyte plastome were found. Three protein-coding genes � chlI, rps4 and rbcL � were used as separated phylogenetic markers or in combined. The results confirmed that one colorless lineage (presented by CCAC 0056, CCAP 977/2a, M2452, M2180) had accelerated evolutionary rates in all gene or/and protein trees. The observations also suggested that chlI gene increased its substitution rate earlier than rps4 and rbcL genes as well as the elevated evolutionary rates could be ordered by chlI > rps4 > rbcL. Although having moderate size (609 bp), rps4 had an evolution rate neither as high as in chlI gene nor as low as in rbcL gene, producing acceptable phylogenetic trees for both nucleotide and protein levels. Therefore, rps4 gene seems to be more suitable protein-enoding plastid gene maker for phylogenies than its sisters, chlI and rbcL genes. The ratio of NNC to NNU in two-fold degenerate NNY codons was calculated for each gene and discussed. It is possible that the shift in codon usage from NNC to NNU did not correlate to the relaxation of functional constraints and/or reduction of gene expression levels. Furthermore, the usage of NNU codons over the NNC in two-fold degenerate NNY codon seemed to be controlled by neutral mutation pressure rather than by selection followed by the gradually acceleration of evolutionary rate. A hypothetical scenario for the relations among the loss of photosynthesis, increasing of substitution rate of interring genes and time of diverging in colorless lineages was discussed

Organelle Genetics in Plants

Chloroplasts in photosynthetic organisms and mitochondria in a vast majority of eukaryotes, contain part of the genetic material of a eukaryotic cell. The organisation and inheritance patterns of this organellar DNA are quite different to that of nuclear DNA. Present-day chloroplast and mitochondrial genomes contain only a few dozen genes. Nevertheless, these organelles harbor several thousand proteins, the vast majority of them encoded by the nucleus. As a result, the expression of nuclear and organelle genomes has to be very precisely coordinated. The selection of experimental and review papers of this book covers a wide range of topics related to chloroplasts and plant mitochondria research, illustrating recent advances and diverse insights into the field of organelle genetics in plants. These works represent some of the latest research on the genetics, genomics, and biotechnology of plant mitochondria and chloroplasts, and they are of significant broad interest for the community of plant scientists, especially for those working in the subjects related to organelle genetic

Tempo and mode of diatom plastid genome evolution

Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Their plastids were derived through secondary endosymbiosis of a red alga. Despite years of phylogenetic research, relationships among major groups of diatoms still remain uncertain. Additional plastid genome (plastome) sequences can not only provide more insight into diatom plastid evolution, but also assess phylogenetic relationships among the major lineages of diatoms. In my dissertation, I have more than doubled the available plastome sequences. My work in the plastome evolution in Thalassiosirales, one of the more comprehensively studied orders in terms of both genetics and morphology, showed highly conserved gene content and gene order within this order. I also documented the first instance of the loss of photosynthetic genes psaE, psaI and psaM in Rhizosolenia imbricata. By extensively sampling the diatoms with critical phylogenetic positions, I presented the largest genome scale phylogeny yet published for diatoms based on 103 shared plastid-coding genes from 40 diatoms and Triparma laevis as the outgroup. The most recent diatom classification posits that there are three major clades of diatoms: Coscinodiscophyceae (informally radial centrics), Mediophyceae (bi- or multipolar centrics), and Bacillariophyceae (pennates). Phylogenetic analysis of plastome data recovered the radial centric Leptocylindrus as the sister group to the remaining diatoms and recovered the polar diatoms Attheya plus Biddulphia in a clade sister to pennate diatoms. Statistical analysis comparing this optimal tree to trees constraining diatoms to the existing classification strongly rejected monophyly for the Coscinodiscophyceae and Mediophyceae. Extensive plastome rearrangements and variable gene content were observed among the 40 diatom species. Astrosyne radiata, recovered on the longest terminal branch, experienced extensive gene loss. The nucleotide substitution rates of plastid protein coding genes were estimated, and their patterns were compared across different gene categories. Relationships between substitution rates and plastome characteristics, such as indels, genome size, genome rearrangement, were examined. The analyses also revealed a strong positive correlation between sequence divergence and gene order change in diatom plastomes.Plant Biolog

Chloroplast Biotechnology in Higher Plants: Expressing Antimicrobial Genes in the Plastid Genome

While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate these environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes which eliminates the threat of transgene escape through pollen. Gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. A bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against plant pathogens and the mycotoxin producing saprophyte Aspergillus flavus

Chloroplast Biotechnology in Higher Plants: Expressing Antimicrobial Genes in the Plastid Genome

While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate these environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes which eliminates the threat of transgene escape through pollen. Gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. A bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against plant pathogens and the mycotoxin producing saprophyte Aspergillus flavus

Expanded inverted repeat region with large scale inversion in the first complete plastid genome sequence of Plantago ovata

Plantago ovata (Plantaginaceae) is an economically and medicinally important species, however, least is known about its genomics and evolution. Here, we report the first complete plastome genome of P. ovata and comparison with previously published genomes of related species from Plantaginaceae. The results revealed that P. ovata plastome size was 162,116 bp and that it had typical quadripartite structure containing a large single copy region of 82,084 bp and small single copy region of 5,272 bp. The genome has a markedly higher inverted repeat (IR) size of 37.4 kb, suggesting large-scale inversion of 13.8 kb within the expanded IR regions. In addition, the P. ovata plastome contains 149 different genes, including 43 tRNA, 8 rRNA, and 98 protein-coding genes. The analysis revealed 139 microsatellites, of which 71 were in the non-coding regions. Approximately 32 forward, 34 tandem, and 17 palindromic repeats were detected. The complete genome sequences, 72 shared genes, matK gene, and rbcL gene from related species generated the same phylogenetic signals, and phylogenetic analysis revealed that P. ovata formed a single clade with P. maritima and P. media. The divergence time estimation as employed in BEAST revealed that P. ovata diverged from P. maritima and P. media about 11.0 million years ago (Mya; 95% highest posterior density, 10.06-12.25 Mya). In conclusion, P. ovata had significant variation in the IR region, suggesting a more stable P. ovata plastome genome than that of other Plantaginaceae species.publishedVersio

Evolution of phage-type RNA polymerases in higher plants

In mono- und eudikotylen Pflanzen kodiert eine Genfamilie (RpoT, RNA-Polymerase des T3/T7-Typs) mitochondriale und plastidäre RNA-Polymerasen (RNAP), die den ungeraden T-Phagen-Polymerasen ähneln. RpoT-Gene von Angiospermen sind gut charakterisiert, während aus tiefer abzweigenden Pflanzenspecies bisher lediglich die Gene aus dem Moos Physcomitrella beschrieben wurden. Um einen Beitrag zur Aufklärung der molekularen Evolution der RpoT-Polymerasen im Pflanzenreich zu liefern und um Erkenntnisse über die potentielle Bedeutung von multiplen Phagen-Typ (RNAP) in Pflanzen zu gewinnen, wurden die RpoT-Gene aus dem Lycophyten Selaginella moellendorffii und aus dem basalen Angiosperm Nuphar advena identifiziert und charakterisiert. Selaginella moellendorffii (Moosfarn)-Trace-Sequenzdaten mit hoher Ähnlichkeit zu RpoT-Sequenzen von Angiospermen wurden benutzt, um das full-length SmRpoT-Gen und die entsprechende cDNA zu isolieren. Die SmRpoT-mRNA ist 3542 nt lang und weist einen offenen Leserahmen von 3006 nt auf, der für ein putatives Protein aus 1002 Aminosäuren mit einer molekularen Masse von 113 kDa kodiert. Das SmRpoT-Gen besteht aus 19 Exons und 18 Introns, die in ihren Positionen mit denen aus den Angiosperm- und Physcomitrella-Genen konserviert sind. Mittels Southernblot-Analyse wurde nachgewiesen, dass S. moellendorffii ein single-copy RpoT-Gen kodiert. Für das N-terminale Transitpeptid von SmRpoT konnte gezeigt werden, dass es bei transienter Expression in Arabidopsis- und Selaginella-Protoplasten den Transport von GFP (green fluorescent protein) exclusiv in Mitochondrien vermittelt. In N. advena wurden mittels Screening einer BAC-Bibliothek drei RpoT-Gene identifiziert. Sowohl die genomischen als auch die cDNA-Sequenzen wurden aufgeklärt. Die NaRpoT-mRNAs kodieren putative Polypeptide von 996, 990 und 985 Aminosären. Alle drei Gene besitzen 19 Exons und 18 Introns, die in ihren Positionen mit denen der RpoT-Gene aus Selaginella und allen anderen Landpflanzen konserviert sind. Die kodierten Proteine weisen auf Aminosäureebene einen hohen Konservierungsgrad auf, einschließlich aller essentiellen Regionen und Aminosäurereste, die für die T7-RNAP bekannt sind. Die N-terminalen Transitpeptide zweier der kodierten RNAP, NaRpoTm1 und NaRpoTm2, vermittelten den Import von GFP exclusiv in Mitochondrien, während die dritte Polymerase, NaRpoTp, in Chloroplasten importiert wurde. Interessanterweise muß die Translation der NaRpoTp-mRNA an einem CUG-Codon initiiert werden, um ein funktionelles Protein mit plastidärem Transitpeptid zu erhalten. Die N. advena RpoTp-RNAP ist somit neben AGAMOUS aus Arabidopsis und der RpoTp-RNAP aus Nicotiana, ein weiteres Beispiel für jene selten vorkommenden pflanzlichen mRNAs, deren Translation exclusiv an nicht-AUG-Codons initiiert wird. Die Rekonstruktion von phylogenetischen Bäumen resultierte in unterschiedlichen Positionen für die Selaginella- und Nuphar-Polymerasen: Im Gegensatz zu der RpoT-Polymerase aus S. moellendorffii und denen aus Physcomitrella, die in den phylogenetischen Analysen Schwesterpositionen zu allen anderen Phagentyp-RNAP der Angiospermen einnehmen, clusterten die Nuphar-RpoTs zusammen mit den deutlich separierten mitochondrialen (NaRpoTm1 und NaRpoTm2) und plastidären (NaRpoTp) Polymerasen. Selaginella kodiert eine einzige mitochondriale RNAP, während Nuphar zwei mitochondriale und eine plastidäre RNAP besitzt. Die Identifizierung einer Plastiden-lokalisierten Phagentyp-RNAP in diesem basalen Eudikotylen, die ortholog zu allen anderen RpoT-Enzymen der Blütenpflanzen ist, läßt darauf schließen, daß die Acquisition einer nukleär kodierten plastidären RNAP, die noch in den Lycopoden fehlt, nach der Trennung der Leucopoden von allen anderen Tracheophyten erfolgte. Eine “dual-targeting” RNAP (mitochondrial und plastidär lokalisiert), wie sie in Eudikotylen, nicht jedoch in Monokotylen vorkommt, wurde weder in Selaginella noch in Nuphar nachgewiesen, vermutlich ist sie ein evolutionäres Novum von eudikotylen Pflanzen wie Arabidopsis.In mono- and eudicot plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species until recently were limited to the moss Physcomitrella. To elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we identified and characterized RpoT genes in the lycophyte Selaginella moellendorffii and the basal eudicot Nuphar advena. Selaginella moellendorffii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3,006 nt, encoding a putative protein of 1,002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. Using Southern blot analysis, it was shown that S. moellendorffii encodes a single RpoT gene. The N-terminal transit peptide of SmRpoT was shown to confer targeting of green fluorescent protein (GFP) exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. In Nuphar advena three RpoT genes were identified by BAC library screening. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those from S. moellendorffii and the RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of GFP exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp polymerase, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. Reconstruction of phylogenetic trees revealed different positions of the RpoTs from the lycophyte Selaginella and the basal eudicot Nuphar. In contrast to the RpoTs of S. moellendorffii and those of the moss Physcomitrella, which are according to the phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. Selaginella encodes a single mitochondrial RNAP, whereas Nuphar harbors two mitochondrial and one plastid phage-type polymerases. Identification of a plastid localized phage-type RNAP in this basal eudicot, orthologous to all other RpoTp enzymes of flowering plants, suggests that the acquisition of a nuclear encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastid RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar or Selaginella suggesting that its occurrence is an evolutionary novelty of eudicotyledoneous plants like Arabidopsis

Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles

The papers included in this Special Issue address a variety of important aspects of Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles, including new advances in the sequencing of both mitochondria and chloroplasts’ genomes using Next-Generation Sequencing technology in plant species and algae including important crop and tree species, in vitro culture protocol, and identification of a core module of genes involved in plastid development. In particular, the published studies focus on the description of adaptive evolution, elucidate mitochondrial mRNA processing, highlight the effect of domestication process on plastome variability and report the development of molecular markers. A meta-analysis of recently published genome-wide expression studies allowed the identification of novel nuclear genes, involved in the complex and still unrevealed mechanisms at the basis of communication between chloroplast and nucleus (retrograde signalling) during plastid development (biogenic control). Finally, an optimized regeneration protocol useful in plastid transformation of recalcitrant species, such as sugarcane, has been reported